Ubiquitin-mediated proteolysis is an important pathway of non-lysosomal protein degradation that controls the timed destruction of many cellular regulatory proteins. Ubiquitin is an evolutionarily highly conserved 76-amino acid polypeptide which is abundantly present in eukaryotic cells. The ubiquitin pathway leads to the covalent attachment of poly-ubiquitin chains to target substrates which are then degraded by a multi-catalytic proteasome complex. In addition to its role in proteasomal degradation of target proteins, the ubiquitin system is also involved in a number of cellular processes unrelated to proteasomal degradation including endocytosis, trafficking in the endosomal system, viral budding, DNA repair, nucleocytoplasmic trafficking and kinase activation.
A number of the steps that regulate protein ubiquitination are known. In particular, initially the ubiquitin activating enzyme (E1) forms a high energy thioester linkage with ubiquitin. Ubiquitin is then transferred to a reactive cysteine residue of one of many ubiquitin conjugating enzymes known as Ubc or ubiquitin E2 enzymes. The final transfer of ubiquitin to a target protein involves one of many ubiquitin protein ligases (E3s).
In eukaryotic cells the ubiquitin proteasome system (UPS) plays an important role in many protein quality control pathways, including the elimination of misfolded proteins from the endoplasmic reticulum (ER) (Hampton, Curr Opin Cell Biol 14, 476-482.2002; Meusser et al., 2005). The UPS dependent degradation of misfolded ER proteins by the so called ERAD pathway (ER-associated protein degradation) adapts cells to stress conditions that would other wise disturb ER homeostasis and cause programmed cell death. An inhibitor of ER-associated protein degradation called Eeyarestatin I (EERI) was recently identified, but the mechanism of its action is unclear (Fiebiger et al., 2004).
To degrade misfolded ER proteins, terminally misfolded polypeptides (both membrane and soluble substrates) are recognized by chaperones, and targeted to the export sites at the ER membrane. Polypeptides are subsequently transferred across the membrane via an unknown conduit to enter the cytosol where they become substrates of the UPS (Friedlander et al., 2000; Ng et al., 2000; Travers et al., 2000). Interestingly, the retrotranslocation pathway can be hijacked by certain viruses to downregulate the expression of correctly folded cellular proteins involved in the immune defense of cells, allowing these viruses to propagate without being detected by the cytotoxic T cells (Lilley and Ploegh, 2005b). For example, either of the two proteins (US11 and US2) encoded by human cytomegalovirus (HCMV) is able to induce rapid dislocation and degradation of newly synthesized MHC class I heavy chains (Wiertz et al., 1996a; Wiertz et al., 1996b).
Because polypeptides can adopt a variety of incorrectly folded states, different misfolded proteins are likely to be distinguished by discrete mechanisms. Genetic studies in yeast have uncovered at least two routes by which misfolded proteins can be selected to undergo retrotranslocation (Swanson et al., 2001; Taxis et al., 2003; Vashist and Ng, 2004). Recent biochemical analyses have identified molecular constituents that account for the mechanistic differences of these pathways. It appears that substrates containing lesions in their luminal domains (ERAD-L substrates) are recognized by chaperones such as Kar2p, Yos9p, and Htm1p/Mn11p, and are targeted to a membrane complex that contains proteins including Der1p, Usa1p, Hrd3p, and the ubiquitin ligase Hrd1p (Carvalho et al., 2006; Denic et al., 2006; Gauss et al., 2006). On the other hand, proteins carrying misfolding signals in their cytosolic domains (ERAD-C substrates) are disposed of by a different set of factors associated with another ubiquitin ligase Doa10p (Carvalho et al., 2006).
When substrates leave the ER, these pathways merge at a highly conserved AAA ATPase (ATPase associated with various cellular activities) termed Cdc48p in yeast or p97/VCP in mammals (Ye, 2006). In mammalian cells, p97 is recruited to the ER membrane via association with two membrane proteins, Derlin and VIMP (Lilley and Ploegh, 2004; Ye et al., 2004; Lilley and Ploegh, 2005a; Ye et al., 2005); whereas in yeast, the link of Cdc48p to the ER membrane is provided by Ubx2p (Neuber et al., 2005; Schuberth and Buchberger, 2005). With the assistance of a dimeric cofactor, Ufd1-Np14, Cdc48p/p97 acts on both ERAD-L and ERAD-C substrates to extract them from the membrane once these substrates are polyubiquitinated (Bays et al., 2001; Hitchcock et al., 2001; Ye et al., 2001; Braun et al., 2002; Rabinovich et al., 2002; Ye et al., 2003; Zhong et al., 2004).
In the next step, substrates dislocated by p97 need to be delivered to the proteasome, which likely occurs in a tightly coupled manner at the ER membrane with the help of some shuttling factors. It was proposed that several ubiquitin binding proteins including a p97-bound ubiquitin ligase Ufd2 and the proteasome-associated factor Rad23 may form a ubiquitin receiving chain to hand over polyubiquitinated substrates to the proteasome (Richly et al., 2005). The degradation of many ERAD substrates also involves a p97-interacting deubiquitinating enzyme (DUB) named ataxin-3 (atx3), which may be part of the substrate delivery system (Wang et al., 2006). The atx3-mediated deubiquitinating reaction appears to act on many misfolded substrates to facilitate their degradation. In the absence of p97-associated deubiquitnation, polyubiquitinated substrates are transferred to the proteasome, but remain intact as proteasome bound species.
Cdc48/p97 not only plays a central role in the ERAD pathway, it is also involved in many other ubiquitin dependent biological processes including cell cycle regulation, transcription control, membrane fusion, protein trafficking et al. (for review see Ye 2005). The p97 ATPase is one of the most abundant proteins in the cytosol (estimated to constitute 1% of the total cytosolic proteins), which is able to interact with various cofactors in a mutually exclusive manner. p97 can act either in conjunction with the proteasome to promote protein turnover, or independently to alter the activity of its substrates. Interestingly, both the proteolytic and non-proteolytic functions of p97 seem to involve ubiquitin one way or the other. Thus, it is generally believed that various p97 cofactors can engage the same ATPase to act on different ubiquitin-modified substrates, which would lead to discrete functional consequences. Intriguingly, several of the identified p97-cofactors are deubiquitinating enzymes, raising the possibility that p97-associated deubiquitination may be essential for its function in various cellular processes.
The development of cancer can depend on the accumulation of specific genetic alterations that allow aberrant cell proliferation, including growth of tumor cells. Ubiquitin is often attached to a substrate as a chain by a process termed polyubiquitination. Polyubiquitination marks the modified protein for degradation through the proteasome. Additionally, ubiquitination can alter the functional state of substrates. p97 is a ubiquitin selective chaperone that regulates many ubiquitin dependent cellular processes critical for cell viability. Compounds that disturb the ubiquitin pathways affect cell growth and other cellular functions. The ubiquitin-proteasome system is recognized to play a large role in tumor biology.
It thus would be desirable to have new compounds that have use in the treatment of undesired cell proliferation, including treatment against cancer cells, as well as for treatment against viral infections. It would be especially desirable to have new compounds that could inhibit p97 functions, including its associated deubiquitinating activities.